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Multiparameter flow cytometric analysis of Granzyme M expression in human peripheral blood leucocytes. Human peripheral blood cells were treated with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) to lyse erythrocytes and fix leucocytes. The leucocytes were washed and then stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with either Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 565571; Left Plot) or Alexa Fluor® 647 Mouse Anti-Human Granzyme M antibody (Cat. No. 566996; Right Plot). A bivariate pseudocolor density plot showing the correlated expression of Granzyme M (or Ig Isotype control staining) versus side light-scatter (SSC) signals was derived from gated events with the forward and side light-scatter characteristics of intact leucocyte populations. Flow cytometric analysis was performed using a BD LSRFortessa™ X-20 Cell Analyzer System.
Two-color flow cytometric analysis of Granzyme M expression in human peripheral blood lymphocytes. Human peripheral blood cells were treated with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) to lyse erythrocytes and fix leucocytes. The leucocytes were washed and then stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with BD Horizon™ BV421 Mouse anti-Human CD3 (Cat. No. 563798/563797) and either Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 565571; Left Plot) or Alexa Fluor® 647 Mouse Anti-Human Granzyme M antibody (Cat. No. 566996; Right Plot). A bivariate pseudocolor density plot showing the correlated expression of CD3 versus Granzyme M (or Ig Isotype control staining) was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ X-20 Cell Analyzer System.
BD Pharmingen™ Alexa Fluor® 647 Mouse Anti-Human Granzyme M
BD Pharmingen™ Alexa Fluor® 647 Mouse Anti-Human Granzyme M
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Preparation And Storage
Recommended Assay Procedures
BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBead to ensure that BD CompBeads are appropriate for your specific cellular application.
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- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
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Companion Products
The 4B2G4 monoclonal antibody specifically recognizes Granzyme M which is also known as GrM, GzmM or Gzm M, as well as, Met-1 serine protease (Hu-Met-1) or Met-ase. Human Granzyme M belongs to a family of effector proteases that include five different granzymes that play key roles in immune responses to pathogens and transformed cells: Granzymes A, B, H, K and M. Granzyme M is a ~ 25-30 kDa neutral serine protease that is encoded by GZMM. Granzyme M participates in innate and adaptive immune responses as it is expressed in the cytotoxic granules of natural killer (NK) cells, NKT cells, cytotoxic αβ T cells and γδ T cells. Granzyme M participates in cell-mediated cytotoxicity by cleaving certain peptide substrates within target cells and promoting caspase activation and subsequent apoptosis. Granzyme M may also display non-cytotoxic functions including the inhibition of viral replication and the promotion of lipopolysaccharide (LPS)-mediated inflammatory responses.
Development References (7)
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Anthony DA, Andrews DM, Chow M, et al. A role for granzyme M in TLR4-driven inflammation and endotoxicosis.. J Immunol. 2010; 185(3):1794-803. (Biology). View Reference
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Bade B, Boettcher HE, Lohrmann J, et al. Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes.. Int Immunol. 2005; 17(11):1419-28. (Clone-specific: Flow cytometry). View Reference
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Bengsch B, Ohtani T, Herati RS, Bovenschen N, Chang KM, Wherry EJ. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patter. J Immunol Methods. 2018; 453:3-10. (Clone-specific). View Reference
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Sayers TJ, Brooks AD, Ward JM, et al. The restricted expression of granzyme M in human lymphocytes.. J Immunol. 2001; 166(2):765-71. (Biology). View Reference
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de Koning PJ, Tesselaar K, Bovenschen N, et al. The cytotoxic protease granzyme M is expressed by lymphocytes of both the innate and adaptive immune system.. Mol Immunol. 2010; 47(4):903-11. (Clone-specific: Flow cytometry). View Reference
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de Poot SA, Bovenschen N. Granzyme M: behind enemy lines.. Cell Death Differ. 2014; 21(3):359-68. (Biology). View Reference
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van Domselaar R, Philippen LE, Quadir R, Wiertz EJ, Kummer JA, Bovenschen N. Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.. J Immunol. 2010; 185(12):7605-13. (Biology). View Reference
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